EA040194B1 - A NEW TARGET FOR THE TREATMENT AND PREVENTION OF DIABETES - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to the field of medicine. More specifically, it relates to diabetes.

Prior Art

Diabetes mellitus or diabetes is a group of metabolic diseases in which a person has high blood sugar levels, either because the pancreas does not produce enough insulin or because the cells do not respond to the insulin it produces.

There are three main types of diabetes:

type 1 results in the body's inability to produce insulin and currently requires the individual to inject insulin or wear an insulin pump;

type 2 is the result of insulin resistance, a condition in which cells do not use insulin properly;

the third is called gestational diabetes and occurs in pregnant women.

Rates of type 2 diabetes have risen markedly since 1960 in parallel with the rise in obesity. As of 2010, there were about 285 million people with the disease, up from about 30 million in 1985. Long-term complications from high blood sugar can include heart disease, stroke, diabetic retinopathy, chronic kidney disease that may require dialysis, and poor circulation in the extremities leading to amputations. A nonketotic hyperosmolar coma may occur.

Hyperglycemia has been reported to be involved in the onset and progressive exacerbation of diabetes mellitus, ie. refers to the theory of glucose toxicity. Namely, chronic hyperglycemia leads to a decrease in insulin secretion and further to a decrease in insulin sensitivity, and as a result, the concentration of glucose in the blood rises in such a way that diabetes mellitus aggravates itself. Therefore, in the treatment of hyperglycemia, the cycle of the aforementioned self-aggravation is interrupted in such a way that prevention or treatment of diabetes mellitus becomes possible.

Unfortunately, existing treatments fail to restore normoglycemia in the long term, as β-cell function declines over time. In addition, at present there is no single drug that can completely change all aspects of this disease.

The progressive nature of type 2 diabetes means that many patients will eventually require a combination of an oral hypoglycemic agent, possibly together with insulin and/or exenatide injections. Antidiabetic agents have been developed to counteract the main mechanisms involved in type 2 diabetes mellitus: insulin resistance (biguanides and thiazolidinediones) and insulin secretion (sulfonylureas, glinides, dipeptidyl peptidase-4 inhibitors, 1 glucagon-like peptide receptor agonists), agents that delay the absorption of glucose in the gastrointestinal tract or promote weight loss, and novel agents that promote the excretion of glucose in the renal tubules. However, most of these drugs have been shown to have detrimental side effects such as weight gain, peripheral edema, or congestive heart failure, and there is a serious problem with the loss of efficacy of these agents on long-term use. Thus, despite a growing number of therapeutic options for glycemic control, there is a need for alternative and improved drugs for the treatment of diabetes and related conditions.

The essence of the invention

The authors of the present invention have identified a new target for the treatment of diabetes, in particular type 2 diabetes. Their groundbreaking finding is ALMS 1 (Ahlström syndrome protein 1), which is involved in insulin regulation of glucose uptake by mature adipocytes through its binding interactions with key molecules involved in glucose regulation. Briefly, they showed that when insulin binds to its receptor, a protein complex (ALMSome) is formed and activated near Alms1, leading to activation of the H ⁺ pump, translocation of the GLUT4 receptor, and glucose uptake by adipocytes. They also showed that in the absence of Alms1, and in consequence of this preventing the assembly of the ALMSome, glucose cannot be transported into cells due to the inability of GLUT4 to fuse with the cell membrane. Therefore, they showed that ALMS1 complexation modulation could be used to regulate glucose transport and thus could be used to bypass insulin resistance and to treat type 2 diabetes.

More specifically, the inventors have identified two proteins involved in the regulation of glucose transport by ALMS1, namely TBC1D4 (a 4th member of the family with the TBC1 domain) and aPKC (PKCa or α-type protein kinase C). More specifically, the binding sites of these two glucose regulating proteins to ALMS1 are so close that simultaneous binding of both proteins is not possible due to steric hindrance. TBC1D4, through its interaction with proteins (ie Rab10, Rab14, etc.) and ALMS1, regulates the translocation of GLUT4 receptors to the cell membrane. On the other hand, aPKC, upon binding to ALMS1, blocks the TBC1D4 binding site and thus negatively regulates the translocation of GLUT4 receptors to the cell membrane, reducing cellular glucose uptake. In addition, they demonstrated that targeting the interaction of ALMS1 and aPKC is sufficient to induce glucose uptake by adipocytes, regardless of the presence of INS. Accordingly, new therapeutic strategies demonstrated in this invention are to increase cellular glucose uptake and reduce hyperglycemia by blocking the binding of aPKC to ALMS1. Most preferably, the binding of aPKC to ALMS1 is inhibited such that the binding of TBC1D4 to ALMS1 is not altered or even enhanced.

Accordingly, the present invention relates to a molecule capable of preventing the binding of aPKC to ALMS1 for use in treating or slowing the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity, and hyperinsulinemia. . It also relates to the use of such a molecule for the preparation of a medicament for treating or slowing the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity and hyperinsulinemia. It also relates to a method for treating or slowing the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity, and hyperinsulinemia in a subject in need thereof, wherein a therapeutically effective amount is administered. a molecule capable of preventing aPKC from binding to ALMS1, thereby increasing insulin-induced glucose uptake. In a preferred embodiment, the molecule does not interfere with the binding of TBC1D4 to ALMS1. Preferably the molecule is selected from the group consisting of peptides or polypeptides or peptidomimetics, antibodies, fragments or derivatives thereof, aptamers, spiegelmers and chemical compounds. More preferably, the molecule is a peptide of less than 50 amino acids, preferably less than 20 amino acids.

In a first preferred embodiment, the molecule is a peptide containing the amino acid sequence of the ALMS1 fragment (SEQ ID NO: 1). Preferably, the molecule is a peptide containing the amino acid sequence of the ALMS1 fragment, including one or more of the residues predicted to mediate interaction with aPKC, in particular one or more of the residues selected from the list consisting of E17, D58, S59, G62, H65, L66, Q736, T737, E738, D828, S829, T1088, D1089, A1169, Q1170, F2882, L2883 and E2884. In the most specific embodiment, the molecule is a peptide containing or consisting of one of the following sequences:

LDSDSHYGPQHLESIDD (SEQ ID NO: 5);

DSHQTEETL (SEQ ID NO: 6);

QQTLPESHLP (SEQ ID NO: 7);

QALLDSHLPE (SEQ ID NO: 8);

PADQMTDTP (SEQ ID NO: 9);

HIPEEAQKVSAV (SEQ ID NO: 10);

SCIFLEQ (SEQ ID NO: 11) and a 6 contiguous amino acid fragment thereof.

In a second preferred embodiment, the molecule is a peptide containing the amino acid sequence of the aPKC fragment (SEQ ID NO: 4). Preferably, the molecule is a peptide containing the amino acid sequence of the aPKC fragment, including one or more of the residues predicted to mediate interaction with ALMS1, in particular one or more of the residues selected from the list consisting of F114, D116 , C118, L121, N138, Q142, I145, P148, G433, E545, S562, S566, F597, D601, W602, K604, E606, G620, T631, V664 and I667.

The present invention also relates to a method for identifying molecules suitable for use in treating or slowing the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity and hyperinsulinemia, in which the ability to molecules to prevent the binding of aPKC to ALMS1 and select molecules capable of preventing this binding. The method may further include testing the ability of the selected molecule to interfere with TBC1D4 binding to ALMS1 and selecting molecules that do not interfere with this binding. Preferably, binding is determined in an insulin responsive cell system. Optionally, binding is determined in the presence and/or absence of insulin.

Another therapeutic strategy identified in this invention is to increase

- 2 040194 cellular uptake of glucose by enhancing the binding of TBC1D4 to ALMS1. Another therapeutic strategy identified in this invention is to increase cellular glucose uptake by upregulating ALMS1 expression.

Thus, the present invention further provides a molecule capable of enhancing the binding of TBC1D4 to ALMS1 or increasing the expression of ALMS1 for use in treating or delaying the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity, hyperinsulinemia and, in particular, type 2 diabetes mellitus. It also relates to the use of such a molecule for the manufacture of a medicament for treating or slowing the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity, and hyperinsulinemia, in particular diabetes mellitus. type 2 diabetes. It also relates to a method for treating or slowing the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity, and hyperinsulinemia, in particular type 2 diabetes mellitus, in a subject, in need thereof, wherein a therapeutically effective amount of a molecule capable of enhancing the binding of TBC1D4 to ALMS1 or increasing the expression of ALMS1 is administered, thereby increasing insulin-induced glucose uptake. In a preferred embodiment, the molecule also inhibits the binding of aPKC to ALMS1.

The present invention also relates to a method for identifying a molecule suitable for use in the treatment of diabetes, which analyzes the ability of the molecule to increase the expression of ALMS1 and select molecules capable of upregulating ALMS1. It also relates to a method for identifying a molecule suitable for use in the treatment of diabetes, in which the ability of the molecule to increase the binding of TBC1D4 to ALMS1 is analyzed and molecules are selected that are capable of increasing this binding parameter. Optionally, the method further comprises determining the ability of the molecule to prevent aPKC from binding to ALMS1, analyzing and selecting molecules capable of preventing this binding.

Detailed description of the invention

The inventors have identified ALMS1 as the missing key player involved in the regulation of insulin-mediated glucose uptake by GLUT4 sorting vesicles in adipocytes.

It is now recognized that even if adipose tissue is responsible for about 20% of glucose absorption, dysfunction in this tissue can lead to diabetes. Thus, any agent capable of regulating insulin-mediated glucose uptake in adipocytes should be able to delay, reverse, or prevent the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity, and hyperinsulinemia.

ALMS1 activity is negatively regulated upon binding to aPKC and upregulated upon binding to TBC1D4. It has also been shown that the binding sites of these two proteins on ALMS1 are so close that simultaneous binding of both proteins is not allowed due to steric hindrance. Thus, this regulatory mechanism is a new target for treating or slowing the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity and hyperinsulinemia, and the inventors propose to use a molecule capable of prevent the binding of aPKC to ALMS1, in these therapeutic indications.

Definitions.

ALMS1, the Alström syndrome protein 1, is a protein encoded by the ALMS1 gene. Mutations in the ALMS1 gene have been found to be responsible for Alström syndrome. They are described in several databases, namely UniProt ID No Q8TCU4; Gene ID No 7840, HGNG ID No 428. Reference sequences are available from Genbank under accession number NM_015120.4 for mRNA and NP_055935.4 for protein. The protein sequence of human ALMS1 is described in SEQ ID NO: 1.

TBC1D4 (4th member of the TBC1 domain family), also currently referred to as As160, putatively acts as a GTPase-activating protein for RAB2A, RAB8A, RAB10 and RAB14. It is described in several databases, namely UniProt ID No 060343, Gene ID No 9882, HGNG ID No 19165. sequences). Isoform 2, which differs from the isoform in the absence of amino acids at positions 678-740, represented in UniProt as No. O60343-2, stimulates translocation of the insulin-induced glucose transporter SLC2A4/GLUT4 to the plasma membrane, thereby increasing glucose uptake. The protein sequence of human TBC1D4 (isoform 1) is shown in SEQ ID NO: 2. The protein sequence of human TBC1D4 (isoform 2) is shown in SEQ ID NO: 3.

- 3 040194

The α-type protein kinase C, also referred to as aPKC, PKC-A, or PKC-a, belongs to a family of serine- and threonine-specific protein kinases that can be activated by calcium and the second messenger diacylglycerol. It is described in several databases, namely UniProt ID No P17252, Gene ID No 9393, HGNG ID No 5578. Reference sequences are described in Genbank with NM_02737.2 for mRNA and NP_002728.1 for protein. The protein sequence of human aPKC is described in SEQ ID NO: 4.

Screening methods.

The present invention relates to an in vitro or ex vivo method for identifying, screening or selecting a molecule capable of preventing aPKC from binding to ALMS1. The method includes determining the effect of the molecule(s) on aPKC binding to ALMS1 and selecting the molecule(s) if aPKC binding to ALMS1 is reduced or prevented. Preferably, the method further comprises determining the effect of the molecule(s) on TBC1D4 binding to ALMS1 and eliminating the molecule(s) if TBC1D4 binding to ALMS1 is reduced or prevented. Optionally, this method may include the step of selecting the molecule(s) if TBC1D4 binding to ALMS1 is increased or enhanced.

The present invention also relates to an in vitro or ex vivo method for identifying, screening or selecting a molecule capable of enhancing or increasing the binding of TBC1D4 to ALMS1. The method includes determining the effect of the molecule on TBC1D4 binding to ALMS1 and selecting the molecule if TBC1D4 binding to ALMS1 is increased or increased. Optionally, the method further comprises determining the effect of the molecule(s) on aPKC binding to ALMS1 and selecting the molecule(s) if aPKC binding to ALMS 1 is reduced or prevented.

In order to determine the effect of a molecule on the binding of aPKC and/or TBC1D4 to ALMS1, any technique known to those skilled in the art may be implemented, in particular any method suitable for determining protein interactions. For example, recombinant or purified native ALMS1 or aPKC can be coupled to a surface plasmon resonance chip and another molecule can be flowed over the chip for binding affinity evaluation, such as in a Biacore device (General Electric, USA). The same approach can be used to measure the binding affinity of ALMS1 and TBC1D4 or ALMS1 and aPKC.

The effect of the molecule(s) on the binding of aPKC and/or TBC1D4 to ALMS1 is determined by measuring the binding of aPKC and/or TBC1D4 to ALMS1 in the absence and presence of the test molecule and by comparing the binding of aPKC and/or TBC1D4 to ALMS1.

In addition, the screening method may include a preliminary step of selecting molecules(s) capable of binding to ALMS1. Indeed, it may be advantageous for the molecule to prevent interactions between ALMS1 and aPKC to act directly at the binding site of ALMS1 to aPKC.

Alternatively, the screening method may include a preliminary step of selecting a molecule capable of binding to aPKC. Indeed, it may also be advantageous for the molecule preventing interactions between ALMS1 and aPKC to act directly at the binding site of aPKC to ALMS1.

In addition, the screening method may include a preliminary step of selecting a molecule capable of binding to TBC1D4.

In a preferred embodiment, for identifying, screening or selecting a molecule capable of preventing aPKC from binding to ALMS1, the screening method of the present invention further comprises determining the effect of the molecule(s), in particular the selected molecule(s), on the binding of TBC1D4 to ALMS1 and selecting the molecule(s). ) if TBC1D4 binding to ALMS1 is not reduced or prevented by the molecule(s). Moreover, the method may include the step of selecting the molecule(s) if TBC1D4 with ALMS1 is increased or enhanced by the molecule(s).

In a preferred embodiment, for identifying, screening or selecting a molecule capable of increasing TBC1D4 binding to ALMS1, the screening method of the present invention further comprises determining the effect of the molecule(s), in particular the selected molecule(s), on aPKC binding to ALMS1 and selecting the molecule(s). ) if aPKC binding to ALMS1 is reduced or prevented by the molecule(s).

Due to the large size of binding partners, in particular ALMS1 and TBC1D4, the inventors prefer to use cell systems for screening methods. Preferably the cellular communication system is an insulin responsive cellular system. For example, the cell system may be selected from a variety of mesenchymal cell lines, mesenchymal stem cells, adipose mesenchymal stem cells, preadipocytes, and adipocytes. Preferably, the cell is a human cell.

Further binding determinations can be made in the presence or absence of insulin. Preferably in the presence of insulin to bind aPKC to ALMS1 and in the presence of insulin to bind TBC1D4 to ALMS1.

- 4 040194

In a first aspect, an immunoprecipitation assay can be performed using ALMS1 as a bait, specifically as detailed in the experimental section. For example, the assay can be performed with cells, in particular adipocytes, cultured in the absence and/or presence of insulin, preferably in the absence of insulin. Test molecules are added to the culture medium. The aPKC is then subjected to immunodetection. Optionally, TBC1D4 can also be subjected to immunodetection. This method is described in detail in the Examples section.

In a preferred embodiment, the amount of aPKC associated with ALMS1 is determined and compared to the amount in the absence of test molecules, in particular in the absence of insulin. If the amount of aPKC associated with ALMS1 decreases in the presence of the test molecule, then the molecule is selected.

The amount of TBC1D4 associated with ALMS1 is determined and compared with the amount in the absence of test molecules, in particular in the presence of insulin or both in the presence and absence of insulin. If the amount of TBC1D4 associated with ALMS1 decreases in the presence of the test molecule, then the molecule is rejected. If the amount of TBC1D4 associated with ALMS1 increases in the presence of the test molecule, then the molecule is selected.

In a second aspect, affinity purification in combination with mass spectrometry can be carried out, in particular, after chemical cross-linking. For example, cells can be grown in a medium lacking methionine and leucine, but including photoactivated methionine and leucine. The cells are then irradiated with UV to stabilize the protein complexes and the protein complexes are analyzed by mass spectrometry.

Other methods are available to those skilled in the art, such as bimolecular fluorescence complementation, tandem affinity purification, and the like. In particular, WO 2012/117245 describes a method for identifying molecules capable of preventing interaction between two proteins: WO 2012/117245 (for identifying small molecules). WO 12174489 also discloses methods for designing molecules suitable for preventing interaction between two proteins.

In addition, suitable molecules can also be designed using molecular modeling. Such methods are, for example, described in detail in the examples section.

In a specific aspect, the present invention relates to the structural homology model of ALMS1 and its use as an in silico method for identifying molecules capable of inhibiting or stimulating ALMSoma function, in particular to inhibit the interaction between ALMS1 and aPKC and/or to increase the interaction between ALMS1 and TBC1D4 .

It also relates to the TBC1D4 structural homology model and its use in an in silico method to identify molecules capable of inhibiting or stimulating ALMSoma function, in particular increasing the interaction between ALMS1 and TBC1D4.

The present invention also relates to a method for identifying, screening or selecting a molecule capable of positively regulating ALMS1 at the level of genes and proteins. The method includes determining the effect of molecule(s) on ALMS1 expression and selecting molecule(s) if ALMS1 expression is increased. In order to determine the effect of a molecule on the expression of ALMS1, any technique known to the person skilled in the art can be implemented.

Various methods known in the art can be used to detect or quantify ALMS1 expression, including sequencing, hybridization, amplification, and/or binding to specific ligands (eg, antibodies). Suitable methods include Southern blotting (for DNA), blotting (for RNA), fluorescent in situ hybridization (FISH), gel migration, ELISA, radioimmunoassays (RIA) and enzyme immunoassays (IEMA).

Increased, increase or enhancement is intended to mean binding increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to binding measured in the absence of the test molecule under the same conditions. Reduced or reduction is intended to mean a reduction in binding of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to binding measured in the absence of the test molecule under the same conditions.

In addition, the screening methods of the present invention may include the analysis of animal models. Test molecules can be introduced into animal models and the effect of the molecules on glucose or diabetes uptake can be evaluated. For example, animal models can be mice or rats with insulin resistance, diabetes mellitus, or hyperglycemia. The effect of the molecule can be assessed by measuring blood glucose levels.

Molecules.

Molecules capable of preventing or blocking the binding of aPKC to ALMS1 can be any ligand capable of binding either aPKC or ALMS1 and thus preventing or blocking the binding of aPKC to ALMS1.

In a first aspect, the present invention relates to a molecule that prevents or blocks

- 5 040194 aPKC binds to ALMS1 by interacting with one or more of the ALMS1 residues selected from the list consisting of E17, D58, S59, G62, H65, L66, Q736, T737, E738, D828, S829, T1088, D1089, A1169, Q1170, F2882, L2883 and E2884. In another aspect, the present invention relates to a molecule that prevents or blocks the binding of aPKC to ALMS1 by interacting with one or more of the aPKC residues selected from the list consisting of F114, D116, C118, L121, N138, Q142, I145, P148, G433 , E545, S562, S566, F597, D601, W602, K604, E606, G620, T631, V664 and I667.

Molecules capable of enhancing or increasing the binding of TBC1D4 to ALMS1 can be any ligand capable of binding either TBC1D4 or ALMS1 and thereby increasing or increasing the binding of TBC1D4 to ALMS1.

In a first aspect, the present invention relates to a molecule that enhances or increases the binding of TBC1D4 to ALMS1 by interacting with one or more of the ALMS1 residues selected from the list consisting of H65, L66 and S2879. In another aspect, the present invention relates to a molecule that enhances or increases the binding of TBC1D4 to ALMS1 by interacting with one or more of the TBC1D4 residues selected from the list consisting of G75, A76, P77, A78, R80, E81, V82 and I83.

The present invention relates to such molecules, a pharmaceutical composition containing such molecules, and the use of such molecules as a drug or for the manufacture of a drug.

These molecules may be peptides or polypeptides or peptide mimetics, antibodies, fragments or derivatives thereof, aptamers, Spiegelmers or chemical compounds. These molecules can be selected using screening methods known in the art, or as described above, and can be designed by any convenient in silico modeling technique.

In a preferred embodiment, the molecule is a peptide or polypeptide. In a preferred embodiment, the peptide may have 5 to 50 amino acids. More preferably it has 5 to 20 amino acids. More preferably the peptide or polypeptide contains less than 50 amino acids, more preferably less than 40, 30, 20, 15 or 10 amino acids.

In a first aspect, the molecule is a peptide or polypeptide containing the amino acid sequence of an ALMS1 fragment (SEQ ID NO: 1). In a preferred embodiment, the molecule is a peptide or polypeptide containing the amino acid sequence of the ALMS1 fragment, including one or more of the residues predicted to mediate interaction with aPKC. In particular, these residues are selected from the list consisting of E17, D58, S59, G62, H65, L66, Q736, T737, E738, D828, S829, T1088, D1089, A1169, Q1170, F2882, L2883 and E2884. More preferably, these residues are selected from the list consisting of D58, S59, G62, H65, L66, Q736, T737, E738, D828, S829, T1088, D1089, A1169, Q1170, F2882, L2883 and E2884. D58, S59, G62, H65 and L66 form the first interaction segment. T737 and E738 define the second segment of the interaction. D828 and S829 define the third interaction segment. T1088 and D1089 define the fourth interaction segment. A1169 and Q1170 define the fifth interaction segment. F2882, L2883 and E2884 define the sixth interaction segment.

In a very specific aspect, the peptide or polypeptide contains or consists of one of the following sequences:

LDSDSHYGPQHLESIDD (SEQ ID NO: 5) targeting the first interaction segment;

DSHQTEETL (SEQ ID NO: 6) targeting the second interaction segment;

QQTLPESHLP (SEQ ID NO: 7);

QALLDSHLPE (SEQ ID NO: 8) targeting the third interaction segment;

PADQMTDTP (SEQ ID NO: 9) focused on the fourth interaction segment;

HIPEEAQKVSAV (SEQ ID NO: 10) focused on the fifth interaction segment;

SCIFLEQ (SEQ ID NO: 11), focused on the sixth segment of the interaction, their fragment containing 6 contiguous amino acids.

In a second aspect, the molecule is a peptide or polypeptide containing the amino acid sequence of an aPKC fragment (SEQ ID NO: 4). In a preferred embodiment, the molecule is a peptide or polypeptide containing the amino acid sequence of the aPKC fragment, including one or more of the residues predicted to mediate interaction with ALMS1. In particular, these residues are selected from the list consisting of F114, D116, C118, L121, N138, Q142, I145, P148, G433, E545, S562, S566, F597, D601, W602, K604, E606, G620, T631, V664 and I667. F114, D116, C118 and L121 may define the first interaction segment. N138, Q142, I145 and P148 may define a second interaction segment. E545, S562 and S566 may define a third interaction segment. F597, D601, W602, K604 and E606 define the fourth interaction segment. V664 and I667 may define a fifth interaction segment.

Optionally, the peptide or polypeptide may include one, two, three, four or five amino acid substitutions compared to the reference sequence, i. sequence SEQ ID NO: 1

- 6 040194 for peptides derived from ALMS1, SEQ ID NO: 4 for peptides derived from aPKC and SEQ ID NO:

or 3 for peptides derived from TBC1D4.

The peptide or polypeptide may further comprise a moiety that facilitates its uptake by the cell or entry into the cell, in particular a PTD (Protein Transduction Domain). A PTD typically contains a defined sequence of 10-20 amino acids (Matsushita and Matsui, (2005), J. Mol. Med. 83, 324328; Vives et al., Biochimic et Biophysica Acta, 2008, 1786, 126-138). PTD is primarily composed of basic amino acids such as arginine or lysine, and typical examples of PTD include arginine-rich peptides such as polyR ₈ (RRRRRRRR) or (RRPRRRPRRPRRPRRP), antennapedia peptide or penetratin such as (RQIKIWFQNRRMKWKK) or HIV-Tat (YGRKKRRQRRR).

In a particular aspect, the molecule is an antibody, fragment, or derivative thereof.

The peptide or polypeptide may contain natural amino acids and/or non-natural amino acids. Non-natural amino acids are intended to refer to an analogue or derivative of a naturally occurring amino acid (i.e. Ala, Val, Gly, Leu, Ile, Lys, Arg, Glu, Gln, Asp, Asn, His, Tyr, Phe, Trp, Ser, Pro, Thr , Cys, Met). They have a modified side chain, such as shorter, longer, or with other functional groups. The D and L isomers are considered, in particular because the D isomers are insensitive to the action of proteases. In addition, modifications to some or all of the peptide bonds are also considered to increase resistance to proteolysis, in particular with (-CO-NH-) with (-CH2-NH-), (-NH-CO-), (-CH2- O-), (-CH2-S-), (-CH2-CH2-), (-CO-CH2-), (-CHOHCH ₂ -), (-N=N-) and/or (-CH=CH -). The peptide may represent a carboxyl C-terminus (-COO ^- ) and an amide terminus (-CONH2). The peptide may also be a D-retro inversopeptide sequence as described herein. The N-terminus may be modified, in particular with an acetyl radical. Optionally, the peptide or polypeptide may be pegylated to improve stability. Alternatively, this peptide can be modified to make it a cross-linked peptide. The term cross-linked peptide as used herein refers to an artificially modified peptide in which the structure is stabilized by one or more artificial molecular bridges (crosslinks) that connect adjacent turns of α-helices in the peptide. Techniques for the preparation of cross-linked peptides have been widely discussed, for example, in Verdine & Hilinski (2012, Methods Enzymol, 503, 3-33), WO 10033617 and WO 10011313, the description of which is incorporated herein by reference).

The present invention also relates to a pharmaceutical composition comprising a peptide as defined above and a pharmaceutically acceptable carrier/excipient. It also refers to a peptide as defined above for use as a medicament, or to the use of a peptide as defined above for the manufacture of a medicament.

In another embodiment, the molecule is an antibody, fragment or derivative thereof. Used in this description, the terms antibody and immunoglobulin have the same meaning and are used in the same way in the present invention. The term antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen-binding site that immunospecifically binds to an antigen. Antibodies include any antibody, preferably monoclonal. They can be, for example, IgG (Immunoglobulin G) or VHH (Camelid antibody heavy chain variable domain). Antibodies, fragments or derivatives thereof include Fab, Fab', F(ab') ₂ , ScFv, (ScFv) ₂ , Dab fragments, hypervariable region (CDR) fragments, linear antibodies, single chain antibody molecules, minibodies, dimers and multispecific antibodies, formed from antibody fragments.

Antibodies, fragments or derivatives can block the interaction between ALMS1 and aPKC. Preferably, if they do not have any effect on the interaction between ALMS1 and TBC1D4 or have an effect that enhances the interaction.

In a first embodiment of the invention, the antibody is specific for ALMS1. In particular, the antibody epitope contains one or more of the ALMS1 residues involved in interaction with aPKC, in particular one or more residues selected from the list consisting of E17, D58, S59, G62, H65, L66, Q736, T737, E738, D828, S829, T1088, D1089, A1169, Q1170, F2882, L2883 and E2884.

Otherwise, the antibody is specific for aPKC. In particular, the antibody epitope contains one or more of the aPKC residues involved in interaction with ALMS1, in particular one or more residues selected from the list consisting of F114, D116, C118, L121, N138, Q142, I145, P148, G433 , E545, S562, S566, F597, D601, W602, K604, E606, G620, T631, V664 and I667.

Such antibodies can be prepared by immunizing non-human mammals with immunogenic peptides or proteins containing one or more residues identified as being involved in the interaction between ALMS1 and aPKC. Alternatively, an antibody library can be generated and screened. The resulting antibodies, fragments or derivatives are then screened for their ability to bind one of the interacting partners and/or their ability to prevent, inhibit or block the interaction between ALMS1 and aPKC. In addition, as previously indicated, antibodies, fragments or derivatives can be further screened for their ability to modulate the interaction between TBC1D4 and

ALMS1 and selected if they increase or enhance the interaction.

Antibodies, fragments or derivatives can enhance the interaction between ALMS1 and TBC1D4.

Preferably they have a blocking effect on the interaction between ALMS1 and aPKC.

In a first embodiment of the invention, the antibody is specific for ALMS1. In particular, the antibody epitope includes one or more of the ALMS1 residues involved in interaction with TBC1D4, in particular one or more residues selected from the list consisting of H65, L66 and S2879.

Otherwise, the antibody is specific for TBC1D4. In particular, the antibody epitope contains one or more of the TBC1D4 residues involved in interaction with ALMS1, in particular one or more residues selected from the list consisting of G75, A76, P77, A78, R80, E81, V82 and I83.

Such antibodies can be produced by immunizing non-human mammals with immunogenic peptides or proteins containing one or more residues identified as being involved in the interaction between ALMS1 and TBC1D4. Alternatively, an antibody library can be presented and screened. The resulting antibodies, fragments or derivatives are then screened for their ability to bind one of the interacting partners and/or their ability to enhance or increase the interaction between ALMS1 and TBC1D4. In addition, as previously indicated, antibodies, fragments or derivatives can be further screened for their ability to modulate the interaction between aPKC and ALMS1 and selected if they reduce or block the interaction.

The production of monoclonal or polyclonal antibodies is well known in the art, and any of a variety of available methods can be used (see, for example, Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)). Methods for obtaining single-chain antibodies (US patent No. 4946778), can be adapted to obtain antibodies to the desired polypeptides. In addition, transgenic mice or other organisms, such as other mammals, can be used to express humanized, chimeric, or similarly modified antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments specifically binding to selected antigens (see, for example, McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10 :779-783 (1992)).

For aptamers and spiegelmers, similar methods can be used to select aptamers and spiegelmers. These methods are well known to those skilled in the art.

As used herein, the term aptamers refers to a nucleic acid molecule or peptide capable of binding ALMS1, aPKC, or TBC1D4. It belongs to a class of molecules that represents an alternative to antibodies in the field of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences capable of recognizing virtually any class of target molecules with high affinity and specificity.

Such ligands can be isolated using systematic exponential enrichment ligand evolution (SELEX) from a library of random sequences as described in Tuerk C. and Gold L., Science, 1990, 249(4968): 505-10. A library of random sequences can be obtained by combinatorial chemical synthesis of DNA. In this library, each element is a linear oligomer, ultimately chemically modified, with a unique sequence. Possible modifications, applications and advantages of this class of molecules have been discussed in Jayasena S. D., Clin. Chem., 1999, 45(9): 1628-50.

Peptide aptamers consist of a fixed-conformation antibody variable region exposed to a protein platform such as E. coli thioredoxin A, which are selected from combinatorial libraries, by dihybrid methods (Colas et al., Nature, 1996, 380, 548-50).

Spiegelmers have been described, for example, in WO 98/08856. They are molecules similar to aptamers. However, Spiegelmers are composed entirely or mainly of L-nucleotides, and not of D-nucleotides, in contrast to aptamers. In other respects, in particular with regard to possible lengths of spiegelmers, the same applies to spiegelmers as described for aptamers.

Chemical compounds refer to molecules of less than about 1500 Da, 1000 Da, 800 Da, or even less than about 500 Da, in particular organic or inorganic compounds. Structural design in chemistry should help find such a molecule. The molecule can be identified using the screening method described in the present invention.

Synthetic compound libraries are commercially available from a number of companies, including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, New Jersey), Brandon Associates (Merrimack, New Hampshire) and Microsource (New Milford, Connecticut). Combinatorial libraries are available or can be prepared according to known synthetic methods. Otherwise, libraries of natural compounds in the form of extracts of bacteria, fungi, plants and animals

- 8 040194 is available from, for example, Pan Laboratories (Bothell, Wash.) and MycoSearch (North Carolina), or can be readily obtained by methods well known in the art.

In addition, natural and synthetically produced libraries and compounds can be further modified using conventional chemical and biochemical methods.

The molecule can be covalently or non-covalently linked to a group that targets appropriate tissues, preferably adipose tissue, or to a group that facilitates the entry of the molecule into cells.

Therapeutic indications.

The authors propose to use the molecules described in this document to increase the uptake of glucose, in particular by adipocytes, whereby the regulation and control of blood glucose levels will be carried out. In this case, the molecules are useful for treating or slowing the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity and hyperinsulinemia.

Diabetes mellitus is characterized by hyperglycemia. More specifically, type 2 diabetes is characterized by hyperglycemia and insulin resistance. Obesity is considered the main cause of type 2 diabetes in people who are genetically predisposed to the disease. Diabetic retinopathy, diabetic neuropathy, diabetic nephropathy are well known disorders associated with diabetes and insulin resistance.

In this case, reducing glycemia by increasing glucose intake can treat or slow the progress or prevent the occurrence of these diseases.

The present invention also relates to molecules of the present invention for use in reducing or stopping insulin doses, when used to treat diabetes in a subject, for using the molecules of the present invention in preparing a medicament for reducing or stopping insulin doses, when used for treatment of diabetes in a subject, or a method of treating diabetes in a subject, wherein a therapeutically effective amount of a molecule of the invention is administered to the subject with a reduced dose of insulin or in the absence of insulin. More generally, it can be used to reduce the dose of antidiabetic drugs.

To treat or cure means that the disease is cured, alleviated or slowed down. This includes preventive or curative treatment. The term treatment means, in particular, the correction, delay or restoration of disturbed glucose homeostasis. The term treatment also means improving glucose uptake (eg, uptake of glucose by adipocytes). In the context of the present invention, the terms blood glucose control or blood glucose control refers to the normalization or regulation of blood or plasma glucose levels in a mammalian subject having abnormal levels (e.g., levels that are below or above a known reference, median, or mean). for the corresponding mammalian subject with normal glucose homeostasis).

The present invention relates to the pharmaceutical or veterinary use of the molecule. Accordingly, the subject may be any mammal, preferably a human, such as an adult or a child. In a particular embodiment, the subject is an obese subject. Optionally, the subject does not have detectable anti-islet antibodies, and ultrasound does not reveal pancreatic disorders. In the context of a veterinary application, the subject may be an animal, preferably a mammal, in particular a pet such as a dog, cat or horse.

The molecules according to the invention can be used in combination with one or more additional active drugs, preferably anti-diabetic drugs, in particular for the treatment or slowing the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity and hyperinsulinemia.

Thus, the present invention also relates to a pharmaceutical composition comprising a molecule of the present invention as well as one or more additional active drugs, preferably an antidiabetic drug.

It also refers to a product or kit containing a molecule according to the invention and one or more additional active drugs, preferably anti-diabetic drugs, in the form of a combined preparation for simultaneous, separate or sequential use, or in the form of a combination preparation, which contains a molecule according to the present invention. and one or more additional active drugs, preferably antidiabetic drugs, for simultaneous, separate or sequential use, in particular for the treatment or slowing the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity and hyperinsulinemia.

It relates to a molecule of the present invention for use in treating or slowing the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity and hyperinsulinemia in combination with one or more additional active drugs, preferably anti-diabetic drugs.

It also relates to the use of a molecule according to the invention and one or more additional active drugs, preferably antidiabetic drugs, for the preparation of a medicament, in particular for the treatment or delay of the progression or delay of the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy. , diabetic nephropathy, insulin resistance, hyperglycemia, obesity and hyperinsulinemia.

Finally, the present invention relates to a method for treating or slowing the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity, and hyperinsulinemia, wherein a therapeutically effective amount of a molecule of the present invention of the invention is administered in combination with a therapeutic or subtherapeutic effective amount of one or more additional active drugs, preferably anti-diabetic drugs. Sub-therapeutic is intended to mean an amount that may be, for example, 90, 80, 70, 60, 50, 40, 30, 20 or 10% of the usual therapeutic dose (in particular, for the same indications and for the same routes of administration).

In particular, the additional active drug is a drug used to treat or slow the progression or delay the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity, and hyperinsulinemia. For example, the additional drug may be an antidiabetic drug such as a hypoglycemic agent or an antihyperglycemic agent. It may be selected from a non-exhaustive list including insulin, metformin, sulfonylureas such as tolbutamide, acetohexamide, tolazamide, chlorpropamide, glibenclamide (also called glibenclamide), glimepiride, glipizide, gliclazide, glycopyramide and glichidone, α-glucosidase inhibitors such as acarbose , miglitol and voglibose, thiazolidinediones such as pioglitazone and rosiglitazone, meglitinide such as repaglinide and nateglinide, incretin mimetics, glucagon-like peptide analogs and agonists such as exenatide, taspoglutide and liraglutide, dipeptidyl peptidase4 inhibitors such as vildagliptin, sitagliptin, saxagliptin , allogliptin and septagliptin, amylin analogs such as pamlintide, SGLT2 inhibitors such as canagliflozin and dapagliflozin, or any combination thereof.

The form of the pharmaceutical compositions, route of administration, dosage and regimen will naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, and so on.

Pharmaceutical or therapeutic compositions of the present invention may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration, etc.

The molecule used in the pharmaceutical composition of the present invention is present in a therapeutically effective amount. The term "therapeutically effective amount" as used herein means that amount of a therapeutic agent administered to a patient that is sufficient to treat diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity, hyperinsulinemia, such as it is defined above.

The pharmaceutical composition containing the molecule is formulated in accordance with standard pharmaceutical practice (Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York), known to those skilled in the art. areas.

For oral administration, the composition may be formulated into conventional oral dosage forms such as tablets, capsules, powders, granules and liquid preparations such as syrups, elixirs and concentrated drops. Non-toxic solid carriers or diluents may be used, which include, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. For compressed tablets, binders, which are agents that impart cohesive properties to powder materials, are also needed. For example, starch, gelatin, sugars such as lactose or dextrose, and natural or synthetic gums can be used as binders. Disintegrants are also needed in tablets to facilitate breaking the tablets. Disintegrants include starches, clays, celluloses, algins, gums, and cross-linked polymers. In addition, lubricants and glidants are also included in the tablets in order to prevent the tablet material from sticking to the surface during production and to improve the flow characteristics of the powder material during production. Colloidal silica is most commonly used as a lubricant, and compounds such as talc or stearic acid are most commonly used as lubricants.

For transdermal administration, the composition may be formulated as an ointment, cream or gel form and appropriate penetrating agents or detergents may be used to facilitate penetration, for example dimethyl sulfoxide, dimethylacetamide and dimethylformamide.

For mucosal administration, nasal sprays, rectal or vaginal suppositories may be used. The active compound may be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycol (carbo waxes), polyethylene sorbitan monostearate, and mixtures thereof with other compatible materials to modify the melting point or dissolution rate.

The pharmaceutical compositions of the present invention may be formulated to release the active drug substantially immediately after administration, or at any predetermined time or period of time following administration.

Pharmaceutical compositions according to the present invention may contain one or more molecules of the present invention associated with pharmaceutically acceptable excipients and/or carriers. These excipients and/or carriers are selected according to the form of administration, as described above.

In a specific embodiment, the pharmaceutical composition of the present invention contains from 0.001 mg to 10 g of the molecule of the invention. In a preferred embodiment, the pharmaceutical composition of the present invention contains from 0.01 mg to 1 g of the molecule of the invention.

Brief description of the drawings

Fig. 1 - metabolic characteristics of mice Alms ^{f z/f z} .

(A) Mean body weight of male WT and Alms ^foz/foz mice (n=6-8 mice per genotype). (B) Photograph of visceral adipose tissue in wild-type and Alms' ^z ' ^z . Scale mark: 25 µm. (C) Insulin tolerance test (ITT) was performed on wild-type mice and Alms ^{f z/f z} mice, and the corresponding histogram shows the area under the curve (AUC) for each genotype (n=6-8 mice per group), p <0.001). (D) Mean body weight of male WT and Alms ^{f z/f z} mice (n=6-8 mice per genotype). (E) Photograph of visceral adipose tissue from corresponding wild-type and Alms ^{f z/f z} mice. Scale mark: 25 µm. (F) An insulin tolerance test was performed on wild-type mice and Alms ^{f z/f z} mice and the corresponding histogram shows the AUC for each genotype (n=6-8 mice per group). *** means p-value <0.001. (G) Immunoblots for the indicated proteins in insulin sensitive tissues from non-obese wild-type mice and Alms ^{f z/f z} . (H) Counting of radioactive signals in various target tissues after injection of radioactive deoxyglucose into wild-type and Alms ^{f z/f z} mice (n=5 mice per genotype). * means p-value=0.05.

Fig. 2 - Silence effect of ALMS1 in mature human adipocytes. (A) Photographs showing the absence of 2-NBDG uptake (green) in control (shCTRL shRNA) or ALMS1 deficient (ALMS1 shRNA) adipocytes silencing mature adipocytes in the absence of INS. (B) Photographs showing no uptake of 2-NBDG in ALMS1 shRNA compared to CTRL shRNA. Nuclei stained with DAPI, DIC: differential interference contrast images. (C) 3D CTRL images of shRNA- or ALMS1 shRNA-mature adipocytes stained for intracellular triglycerides (TG), plasma membrane red (PM) and nuclei blue (DAPI). (D) Measurements of fluorescence levels correlated with the amount of intracellular Tg in mature adipocytes (n=16 wells for each condition measured), *p-value=0.05. (E-F) Immunodetection of AKT and pS473-AKT in CTRL shRNA- and ALMS1 shRNA-treated mature adipocytes in the presence and absence of INS. (G) 3D images of CTRL shRNA- and ALMS1 shRNA-mature adipocytes showing cellular localization of insulin receptor (IR-red) and GLUT4 (green) in the absence of Ins. Cross-sectional images showing the dynamics of GLUT4 localization in the absence of Ins. (H) After 30 min of INS stimulation. (I) and with 30 min of INS stimulation, followed by a 2-hour absence of INS. (J) in CTRL shRNA- and ALMS1 shRNA-mature adipocytes. Scale mark: 25 µm in A, B, C and 5 µm in GJ.

Fig. 3 - predicted sites of interaction on the ALMS1 protein and modeling of its partner TBC1D4. (A) Predicted 3D structure of the ALMS1 protein with helices and loops. (B) Predicted 3D structure of the ALMS1 protein with potential interaction sites represented by red dots. (C) The primary sequence of the TBC1D4 protein with the indicated localization of binding sites or interacting domains. (D) Predicted 3D structure of the TBC1D4 protein.

Fig. 4 - ALMS1 is required for cell directed migration of TBC1D4. (A) In silico predicted 3D structure showing the spatial interaction between ALMS1 and TBC1D4 with an enlarged view of the interaction site highlighting the predicted interactions

- 11 040194 amino acid residues (L66, Y61 and S2879) of the ALMS1 protein. (B) 3D image of immunostained mature adipocytes showing colocalization of TBC1D4 (green) and ALMS1 (red). Nuclei are stained with DAPI (blue). (C-D) Immunoblots of indicated proteins on cell lysates (50 μg total protein loaded per lane) of CTRL shRNA- and ALMS1 shRNA-mature adipocytes treated with or without insulin. 3D images of immunofluorescence experiments performed on either CTRL shRNA-, ALMS1 shRNA-, or TBC1D4 shRNA-mature adipocytes showing cellular localization of GLUT4 in the absence of insulin (-INS) (E) or in the presence of INS. (F). RM: plasma membrane, nuclei stained with DAPI. 3D images of immunofluorescent experiments performed on either CTRL shRNA or ALMS1 shRNA adipocytes demonstrating cellular localization of GLUT4 (green) and TBC1D4 in the absence of INS (G) or 30 min after INS treatment. (H) Scale marks: 10 µm.

Fig. 5 - TBC1D4 is not the only ALMS1 interaction partner that plays a role in adipocyte biology. (A-C) Photographs showing 2-NBDG uptake in either CTRL shRNA-, ALMS1 shRNA-, or TBC1D4 shRNA-deprived adipocytes after 30 min of INS stimulation. (D-F) 3D images obtained using INS-stimulated non-permeabilized mature adipocytes followed by immunodetection of membrane-bound GLUT4 (green). The plasma membrane (PM) was stained with Image-iT (red) and the nuclei were stained with DAPI. (G) Immunoblots of 2 proton pump subunits (ATP6V0D1 and ATP6V1A) identified by mass spectrometry in IP experiments using ALMS1 as bait (Fig. S4), aPKC, GLUT4, and β-tubulin in cell extracts from white adipose tissue (WAT) and kidney, 50 µg total protein loaded per lane. (H) Photograph of a positive Duolink signal detected in adipocytes using antibodies against ALMS1 and ATP6. (I) Immunofluorescence images demonstrating cellular localizations of ATP6V0D1 and ALMS1 and pooled image in mature adipocytes under INS stimulation. (J) In silico predicted TBC1D4 (red) and PKC (yellow) binding sites that are only 20 A apart in the ALMS1 3D structure. (K-L) Immunodetection of aPKC, TBC1D4 and α-actinin in immunoprecipitates using ALMS1 as a bait in adipocytes cultured in the absence or presence of INS.

Fig. 6 - restoration of acidification in ALMSl-deprived adipocytes restores glucose absorption. (A-B) Interval burst images were performed on either control or ALMSI-deprived adipocytes stained with acridine orange and stimulated with INS. (C-D) Interval burst images were performed on either control or ALMSl-deprived adipocytes stained with acridine orange and stimulated with the electrically neutral K+/H+ exchange ionophore, nigericin (NIG). (E) Top to bottom: Scanning electron microscopy (SEM) images of control adipocytes stimulated with either no INS, INS, or NIG. White arrows show swollen vesicles. (F) Transmission electron microscopy (TEM) images corresponding to those presented in (E) demonstrate the fusion of vesicles with the plasma membrane in the presence of INS and NIG. (G) From top to bottom: SEM photographs of ALMSI-deprived adipocytes stimulated with either no INS, INS, or NIG. (H) TEM images corresponding to those presented in (G), demonstrating the fusion of vesicles with the plasma membrane only in the presence of NIG. (I) Photographs showing the intracellular content of 2-NBDG (green) in control mature adipocytes either in the absence of INS (upper panel) or after 30 min of INS stimulation (middle panel) or after 30 min of NIG stimulation (lower panel). (J) Photographs demonstrating the intracellular content of 2-NBDG (green) in ALMSl-deprived mature adipocytes either in the absence of INS (upper panel) or after 30 min of INS stimulation (middle panel) or after 30 min of NIG stimulation (lower panel) . Scale mark: 20 µm except for F and H: 500 nm.

Fig. 7 - directed migration of GLUT4 requires the ALMSoma protein complex. (A) 3D images obtained using non-permeabilized fixed mature adipocytes stimulated with NIG followed by immunodetection of membrane-bound GLUT4 (green). The plasma membrane (PM) was stained with Image-It (red) and the nuclei were stained with DAPI. (B) Photographs showing intracellular TG content 24 h after NIG treatment. (C) Schematic representation of the cellular localization of ALMSl and partner protein in the absence of INS stimulation in a mature adipocyte. (D) Schematic representation of the dynamics of ALMS1 and protein partners after INS stimulation in a mature adipocyte.

Fig. 8 - Glucose uptake is initiated in the absence of INS via specific interference with the aPKC binding site in the ALMSome. (A) Photographs showing 2NBDG uptake in the presence or absence of INS in adipocytes infected with either CTRL lentiviral particles or aPKC domain lentiviral particles. (B) Quantification of the intracellular glucose analogue 2-NB in the presence of either CTRL lentiviral particles or aPKC domain lentiviral particles (n=8 per group).

- l2 040194

Fig. 9 depicts the min-aPKC-FLAG construct in adipocytes. Upper panel: immunodetection of min-aPKC-FLAG using an anti-FLAG antibody in mature adipocytes 48 h after lentivirus infection. Panels 2 and 3: 3D image of adipocytes showing perinuclear localization of min-aPKC-FLAG. Last panel: Schematic representation of the experimental approaches used to evaluate the effect of min-aPKC-FLAG on glucose uptake.

Examples

Alström syndrome (ALMS) is a rare autosomal recessive disorder characterized by several clinical features including obesity and early onset of diabetes. This is due to a mutation in the gene encoding the 460 kDa protein ALMS1.

Until now, the function of the ALMS1 gene and how it causes the Alström syndrome phenotype has been unknown, research on its function is hampered by the extremely large size of the encoded protein and its low levels of expression.

Alström syndrome (ALMS) is a rare monogenic childhood obesity syndrome for which there is only one causative mutant gene identified to date, the ALMS1 gene. ALMS is classified as a member of the ciliopathy disorders that include Bardet-Biedl syndrome, a group of syndromic disorders arising from mutations in a large number of different proteins that together play an important role in primary cilium function. Alms1 encodes the 461 kDa protein ALMS1, which was originally described as having a purely centriolar localization, although more recent data have also suggested a cytoplasmic localization of ALMS1.

ALMS is clinically identified with a cumulative multisystem phenotype that is thought to reflect ubiquitous tissue expression of ALMS1, closely mimicking many of the phenotypic features of BBS. Common clinical features of ALMS include retinal degeneration, hearing loss, childhood obesity, early onset type 2 diabetes mellitus (T2DM), dilated cardiomyopathy, renal and hepatic dysfunction, hypothyroidism, short stature, hyperlipidemia, and organ fibrosis. Children with ALMS develop obesity in early childhood, which is associated with early onset T2DM at about 16 years of age, with a much higher overall prevalence of early onset T2DM in ALMS than seen with other childhood obesity syndromes giving a similar body mass index (BMI). including BBS. The reason for this propensity for T2DM in children with ALMS, which is not proportional to their degree of obesity, remains unclear.

The present inventors investigated the role of the ALMS1 protein in the adipogenic differentiation process and found that the expression levels of the ALMS1 protein increase during adipogenesis. Suppression of ALMS1 in adipogenic differentiating mesenchymal stem cells inhibits anti-adipogenic cascades, but surprisingly not in favor of adipogenesis.

In addition, the authors showed that the ALMS1 protein complex is also required in mature adipocytes for efficient retention of GLUT4 in its insulin-responsive compartment and for its ability to fuse with the plasma membrane in response to insulin stimulation. Inactivation of ALMS1 reduces the amount of glucose that mature adipocytes can take up when stimulated with insulin, thus contributing to hyperglycemia and the onset of diabetes.

Previous studies in the Alms1 ^foz/foz spontaneous mutant and the Alms1 knockout mouse model of ALMS generated by insertion into the gene confirmed that these mice, like malnourished children, develop obesity in early adolescence due to hyperphagia and also exhibit impaired glucose tolerance, hyperinsulinemia, and islet hypertrophy consistent with severe insulin resistance, although the tissue origin or mechanism for this insulin resistance has not been previously characterized. Previously published in vitro studies in the murine fibroblast cell line 3T3-L1 showed that inhibition of ALMS1 gene expression resulted in a moderate impairment of adipogenesis, but it was reported that there was no effect on insulin signaling in the resulting mature adipocytes, as measured by insulin-mediated phosphorylation of AKT. These data deviate from the invention presented herein, in which Alms1 does play a critical, hitherto unknown role in insulin signaling and in GLUT4-mediated glucose transport.

Indeed, despite previously published conflicting data, the authors, by carefully studying the phenotype of the ALMS Fat Aussie (Alms1 ^foz/foz ) mouse model, determined that insulin resistance in this model precedes rather than follows the development of obesity. In addition, they identified adipose tissue as a specific site governing insulin resistance and subsequent development of glucose tolerance and T2DM in ALMS. They confirmed that the insulin signaling pathway in Alms1 ^foz/foz adipocytes was intact all the way down to the phosphorylation of TBC1D4, the last known member of the insulin-mediated glucose uptake pathway, and then, in a subsequent series of studies, they identified a protein complex they called the ALMSome, consisting of several key proteins that bind to ALMS1 and which together are required for the attachment and fusion of GLUT4 vesicles to the plasma membrane (PM) of adipocytes in response to an insulin signal. ALMSoMa thus represents a hitherto unknown last step in insulin-mediated glucose uptake by adipocytes, with insulin resistance in ALMS due to impaired ALMSOma function leading to failure of GLUT4-mediated membrane fusion and thus blocking glucose transport in adipocytes.

Example 1 Alms1 ^foz/foz mice show strong specific insulin resistance in adipose tissue even in the absence of obesity.

Keeping animals.

Alms1 ^foz/foz and Alms1 ^+/+ (wild type) littermates were maintained in C57BL/6J environment in a vivarium with a 12-hour light/dark cycle. Mice had free ad libitum access to water and either a normal diet containing 5.4% fat with an energy content of 12 MJ/kg (Gordon's pellets for keeping mice and rats, Gordon's specialty stockfeeds, Australia) or a diet with a high content of fat (HFD) containing 23% fat, high levels of simple carbohydrates, 0.19% cholesterol, with an energy content of 20 MJ/kg (SF03-020, Specialty feeds, Australia). For PCR genotyping, primers flanking the foz mutation were used: forward ACA ACT TTT CAT GGC TCC AGT (SEQ ID NO: 13); reverse TTG GCT CAG AGA CAG TTG AAA (SEQ ID NO: 14).

Six-month-old obese and young (<60 days of age) non-obese Alms1 ^foz/foz mice and wild-type (WT) littermates were used to investigate the primary metabolic disturbances leading Alms1 ^foz/foz A mice to develop T2DM. Six-month-old Alms1 ^foz/foz mice were obese with a mean body weight of 45.5±1.7 g compared to 26.4±1.3 g for wild-type littermates (Fig. 1A) and were previously shown to have hyperglycemia. fasting and impaired glucose tolerance with elevated HOMA scores. The Insulin Tolerance Test (ITT) showed that, in contrast to WT (FIG. 1B) and heterozygous litter mates, glycemia in obese Alms1 ^foz/foz mice was refractory to insulin administration (FIG. 1B), even when administered doses of insulin reached 20 U/kg (Fig. 1C). Obesity in the Alms1 ^foz/foz mice was due to severe adipocyte hypertrophy (FIG. 1B) rather than the adipocyte hyperplasia typically seen in obese BBS mice. In order to determine which primary defect causes impaired glucose tolerance in Alms1 ^foz/foz mice, young lean Alms1 ^foz/foz mice were studied in order to remove the confounding effects of obesity on insulin responsiveness. At 2 months of age, WT and Alms1 ^foz/foz males had a similar mean body weight of ~24 g (Fig. 1D). ITT in these mice showed that young, non-obese Alms1 ^foz/foz males nevertheless already show significantly reduced insulin responsiveness (FIG. 1E) consistent with pre-obesity insulin resistance in this model. Immunodetection of IRAP, Akt, p-AKT, GLUT4, C/EBP-α and GAPDH performed on insulin-sensitive tissues, namely heart, liver, skeletal muscle and white adipose tissue (WAT), from 6-month-old non-fasted Alms1 ^{foz/ foz} and WT showed no significant differences in protein levels, except for a consistent increase in p-AKT relative to total AKT in WAT, consistent with a paradoxical increase, not decrease, in activation of upstream insulin signaling pathway participants in Alms1 ^foz/foz glucose intolerant mice. (Fig. 1G). In order to determine which tissues, individually or collectively, may be the main source of insulin resistance observed in Alms1 ^foz/foz mice, the tissue distribution of insulin-mediated deoxyglucose (DOG) uptake was compared in WT and Alms1 ^foz/foz mice. This confirms that severely impaired DOG uptake was limited by WAT from Alms1 ^foz/foz mice, with a compensatory increase in DOG uptake in insulin-responsive gastrocnemius and soleus muscles compared to wild-type mice.

These studies show that although Alms1 ^foz/foz mice become obese and develop progressive T2DM with age, the main underlying defect contributing to insulin resistance and hyperglycemia is the impairment, in the absence of functional ALMS1, of glucose uptake by adipose tissue in response to insulin signaling. and this defect precedes the development of obesity.

Example 2 Alms1 silencing in human adipocytes blocks glucose uptake due to disruption of GLUT4 cell sorting.

Materials. From Molecular Probes, Invitrogen: Acridine Orange, Image-iT® LIVE Plasma Membrane and Nuclear Staining Labeling Kit, 2NBDG (2-(N-7-nitrobenz-2-oxa-1,3-thiadiazole- 4-yl)amino)-2-deoxyglucose), Hoechst 33258 and Cell Light™ Early Endosomes-RFP * BacMam 2.0*; Part #: A3568, I34406, N13195, H3569, and C10587. From Lonza: AdipoRedTM Assay Reagent (Catalog #: PT-7009). Lentiviral particles from Santa Cruz Biotechnology, Inc.: ALMS1 shRNA lentiviral particles (h), TBC1D4 shRNA lentiviral particles (h), and lentiviral particles-A with shRNA control; Part #: SC72345-V, SC-61654-V and SC-108080 respectively. From Tocris Biosciences: Nigericin Sodium (Cat #: 4312).

biochemical tests. Mice were tested for insulin resistance using an insulin tolerance test (ITT) and a glucose tolerance test (intraperitoneal IPGTT). For ITT, mice were fasted for 4 h, with no access to food, but with free access to water. Mice were weighed and insulin (Humulin R., Eli Lilly, USA) was injected intraperitoneally at 0.75 U/kg body weight in 0.9% saline injection (Pfizer, USA). Blood was obtained from the tail and plasma glucose was determined for each mouse using a glucometer (Optium Xceed, Abbott, USA) and blood glucose test strips (Optium point of care, Abbott, USA) at times 0, 15, 30, and 60 minutes after insulin injection. For IPGTT, mice were fasted for 18 hours and injected with 2 mg/g body weight of D-glucose (Analar, VWR, USA) in 0.9% saline injection. Plasma glucose levels were determined for each mouse with a tail vein sampling glucometer at 0, 15, 30, 60, and 120 minutes after glucose injection. To measure plasma insulin, blood was taken from awake animals by means of a buccal haemorrhage. After collection, blood samples were kept on ice and centrifuged at 17,000 g, 10 min at 4°C. Insulin levels were analyzed using a commercial ultra-sensitive mouse insulin ELISA kit (Crystal Chem Inc., USA). A homeostatic model for assessing insulin resistance (HOMA-IR) was calculated using fasting insulin and fasting glucose levels in an individual mouse. The following formula was used: HOMA-IR=[fasting glucose (mg/dl) fasting quinsulin (μU/ml)]/405.

Cell culture. Human white visceral preadipocytes (Catalog #: C-12732; PromoCell) and human mesenchymal stem cells (Catalog #: C-12974; PromoCell) were purchased from healthy bone marrow. Preadipocytes were plated according to the manufacturer's protocol and cultured in Preadipocyte Growth Medium (Catalog #: C-27410; PromoCell) until confluent. One day prior to the induction of terminal adipogenesis, cells were infected with specific lentiviral particles consisting of a pool of 3 target-specific shsRNA constructs purchased from Santa Cruz Biotechnology, and the following day, adipogenic differentiation was induced by changing medium to preadipocyte differentiation medium (Catalog # C- 27436; PromoCell) for 2 days. After the differentiation step, the medium was finally changed to Adipocyte Nutrition medium (Catalog # C-27438; PromoCell). For the insulin-free culture, Adipocyte Basal Medium (Catalog #: C-2431; PromoCell) without insulin was supplemented with 5 g/l deoxyglucose, 8 μg/ml d-biotin, 400 ng/ml dexamethasone. For hMSCs, they were cultured in mesenchymal stem cell growth medium (catalog #: C-28010; PromoCell) until confluent. hMSCs were transfected with specific siRNAs as described above and lipogenic differentiation was induced the next day by medium change to MSC Adipogenic Differentiation Medium (Cat #: C28011; Promocell).

RNA isolation, cDNA synthesis, quantitative PCR and Taqman. Total RNA was obtained from various tissues and cells using the RiboPure™ Kit (Cat. #: AM1924; Ambion) followed by DNase with TURBO DNA-free™ (Cat. #: AM 1907; Ambion). RNA integrity was assessed by gel electrophoresis and RNA concentration was determined using an Eppendorf Biophotometer Plus with a Cell Hellma® Tray (Cat #: 105,810-UVS; Hellma). Reverse transcription of 1 μg of total RNA into cDNA was performed using the BioRad iScript™ cDNA Synthesis Kit (Cat #: 170-8891; BioRad). Real-time quantitative PCR amplification was performed in real time on a BioRad CFX96™ system using an IQ™ SYBR® Green Supermix (Cat. #: 170-8886; BioRad) and a primer set optimized for test targets for real-time PCR. time-based SYBR Green for real-time PCR. Taqman analysis was performed with specific gene analysis using TaqMan® Fast Advanced Master Mix (Cat. #: 4444557; Applied Biosystems). Normalized fold expression of the target gene was calculated using the comparative threshold cycle (Ct) method, normalizing the target mRNA Ct to that of GAPDH using CFX Manager Software Version 1.5 and checked using the Lin-Reg program.

Western blots and immunofluorescent microscopy. Male Alms1 ^foz/foz and wild-type littermates were anesthetized. The following insulin sensitive tissues: liver, heart, muscle and adipose tissue were harvested and directly placed in RIPA buffer (Tris 50 mM, NaCl 150 mM, 0.1% SDS, 1% Triton-X100) supplemented with a complete cocktail of mini- proteases and a cocktail of phosphatase inhibitors PhosSTOP (Roche, Switzerland). Samples were sonicated and centrifuged for 30 min at 17000G, 4°C for 30 min. The protein concentration was analyzed using BCA analysis (Thermo Fisher Scientific, USA). Cellular proteins from cells were obtained by precipitation with trichloroacetic acid and immunoblot analyzes were performed using 30-50 μg of total protein. Specific antibody binding was visualized with SuperSignal® West Femto Maximum Sensitivity Substrate (Cat #: Lf145954, Pierce) on a BioRad Versadoc™ Imaging System or ImageQuant LAS 4000 Imager (GE Healthcare, UK). Ponceau S-stained non-specific proteins were used as loading controls to normalize the signal obtained after specific immunodetection of the protein of interest using the Bio-Rad Quantity One program. For immunofluorescence experiments, cells were seeded in the per

- 15 040194 manox 8-wells Lab-Tek II Chamber Slide (Part #: 177445; Nunc). Cells were processed as indicated. Cells and tissue cryosections were then processed for protein detection after fixation with methanol and permeabilization with 0.1% Triton X-100. Slides were mounted for detection using Vectashield Mounting Medium (Catalog #: H-1200; Vector Laboratories). To view membrane-associated proteins, cells were fixed in formalin for 15 min and directly blocked, and then immunostained and imaged using a Zeiss AxioImager Z2 upright microscope. Image analysis, 3D reconstruction and time lapse experiments and endosome tracking experiments were performed either with the Zeiss AxioVision software with appropriate Zeiss 3D and Tracking modules or with the Zeiss Zen 2012 imaging platform.

Fluorescence measurement. Preadipocytes were cultured in a 96-well plate and 12 wells were infected with either ALMS1 shRNA lentiviral particles or shRNA CTRL lentiviral particles and differentiated the next day into mature adipocytes. After 3 weeks, intracellular triglycerides were stained with AdipoRed according to the manufacturer's procedure and fluorescence measured on a Tecan Infinite 200 QUAD4 monochromator (Tecan, Lyon, France) at 520 nm. The data obtained were then analyzed using Tesap Magellan Data Analysis software using unstained adipocytes as a control.

Coimmunoprecipitation experiments. For co-immunoprecipitation experiments, we used the Dynabeads® Antibody Coupling kit (Cat. No.: 143.11D, Invitrogen) in combination with the Dynabeads® co-immunprecipitation kit (Cat. No.: 143.21D, Invitrogen). hMSCs were cultured to confluency and adipogenic differentiation was induced by medium change. 7 days after medium change initiated adipogenic differentiation, adipocytes were cultured with or without Ins 24 h prior to lysis, then lysed under native conditions and used according to the kit. After immunoprecipitation and bead release, samples were loaded onto NuPAGE 38% TrisAcetate gel (catalog number: EA0375BOX, Invitrogen), with Hi Mark™ Prestained HMW Protein Standard (Cat#: LC5699, Invitrogen).

Protein preparation and identification by mass spectrometry. Hydrolysis in gel. The gel hydrolysis procedure was performed as described by Rabilloud et al. (link). Preparation of gel pieces prior to hydrolysis with trypsin was carried out using a liquid processing robot (QuadZ215, Gilson International, France). Briefly, the gel strips were washed alternately with 100 μl of 25 mM NH ₄ HCO ₃ and then with 100 μl of 50% acetonitrile (ACN) (3 min washes with shaking and the liquid was decanted before adding the next solvent). This humidification/dehydration cycle was repeated twice and the gel pieces were dried for 20 min before reduction (10 mM DTT/25 mM NH4HCO3 buffer at 56°C for 45 min) and alkylation (25 mM iodoacetamide/25 mM NH4HCO3 buffer for 45 min , at room temperature). Thereafter, the spots on the gel were again washed with 3 cycles of 25 mM NH ₄ HCO ₃ /ACN in turn. After a 20 min drying step, the gel pieces were rehydrated with three volumes of trypsin (Promega, V5111) 12.5 ng/µl in 25 mM NH4HCO3 buffer (freshly diluted) and incubated overnight at room temperature. The trypsinized peptides were extracted from the gel by vigorous shaking for 30 min in an adapted volume of 35% H ₂ O/60% ACN/5% HCOOH and a 15 min sonication step.

MALDI-TOF (/TOF) mass spectrometry and database search. MALDI measurement was performed on an Autoflex III SmartBeam (Bruker-Daltonik GmbH, Bremen, Germany) laser desorption/ionization array time-of-flight mass spectrometer (MALDI-TOF TOF) used in reflex positive mode. A Prespotted Anchorchip target (PAC system from Bruker Daltonik, Technical Note TN011) with an HCCA array was used to analyze tryptic hydrolysates. Peptide mass fingerprinting (PMF) data and peptide fragment fingerprinting (PFF) data were combined using Biotools 3.2 software (Bruker Daltonik) and the intranet version was transferred to the MASCOT search engine (Matrix Science, London, UK). Variable modifications (N-terminal protein acetylation, methionine oxidation, and cysteine ureamethylation) and one trypsin-missing cleavage were taken into account and the peptide mass error was limited to 50 ppm. Proteins were identified using a data search function against a non-redundant NCBI protein sequence database and then against a database limited to human sequences. In all outcomes, the probability scores were higher than the score set as significant with a p-value of 0.05. NanoLC-MSMS mass spectrometry and database search. For nanoLC-MS/MS analysis, the peptides were transferred into glass inserts compatible with the LC autosampler system (nanoLC-U3000, Dionex, USA). The LC system was connected to an ESI-Q-TOF mass spectrometer (MicroTOFQ-II, Bruker, Germany). The method consisted of a 60 min run at a flow rate of 300 nl/min using a gradient of two solvents: A (99.9% water: 0.1% formic acid) and B (99.92% acetonitrile: 0.08% formic acid) . The system includes: a 300 µm X 5 mm PepMap C18 column used for peptide preconcentration and a 75 µm X 150 column

- 16 040194 mm C18 used to elute the peptides. The TOF analyzer was calibrated every day: data was acquired and processed automatically using Hystar 2.8 and DataAnalysis 2.6 software. Sequential searches first against the NCBInr database and then against a human limited database were performed for each sample using local Mascot 2.2 (MatrixScience, UK) and ProteinScape 2.0 (Bruker, Germany). The relative number of false positive signals (FPR) for protein identification was assessed using a database of reverse traps (reverse decoy): protein validation was performed using an FPR below 1%. In addition, proteins identified by only 1 peptide were checked manually: MS/MS spectra were checked according to the usual fragmentation rules.

In situ close ligation analysis (PLA). The Duolink in situ PLA kit with anti-mouse PLUS probe and anti-rabbit MINUS probe (Cat #: 90701 and 90602; OLink Bioscience) was used in combination with the appropriate primary antibodies according to the manufacturer's protocol. Human primary preadipocytes and mature adipocytes were cultured in an 8-well LabTek II chamber slide (Nunc) and processed as for immunofluorescence microscopy prior to the initial antibody incubation step. After washing, cells were decorated with PLA PLUS and MINUS probes (1:20 dilution) for 2 h at 37°C.

Hybridization and ligation of the probes, amplification and final washing of the SSC were performed according to the manufacturer's protocol. Fluorescence transfer based on protein-protein interactions was visualized using a Duolink 613 detection kit (OLink Bioscience) and imaged.

Statistics. Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software, Inc., USA). The results are presented as means ± standard deviation. The significance of the results was determined using a two-tailed Student's t-test or a non-parametric Mann-Whitney U-test for statistical comparison of BMI and AUC data. The value of P<0.05 was taken into account in determining the statistical significance and marked with an asterisk.

Using primary human preadipocytes as an in vitro model, the authors localized ALMS1 predominantly in the cytoplasmic rather than, as previously reported, in the centrosomal pool. ALMS1 was silenced during adipogenesis, and although there was a significant decrease in the anti-adipogenic factor Pref-1, no significant differences were found in the expression levels of pro-adipogenic transcription factors such as cEBPs and PPARy.

Following ALMS1 silencing in 2-week-old mature adipocytes, glucose uptake capacity was assessed using a labeled glucose analog (2-NBDG). In the absence of insulin stimulation, 2-NBDG uptake was not detected in ALMS1-silencing and control mature adipocytes (FIG. 2A). On the other hand, insulin stimulation results in increased 2-NBDG uptake in control mature human adipocytes (FIG. 2B, upper panel), but not in ALMS1-silencing cells (FIG. 2B, lower panel). In addition to reducing glucose uptake in ALMS1-silencing adipocytes, we observed a decrease in intracellular triglycerides (TG) in these cells as early as a week later (Fig. 2C-D). Of note, this reduced glucose uptake in ALMS1-deficient adipocytes was not associated with a decrease in AKT phosphorylation downstream of the insulin signaling pathway, as pS473-AKT levels after 30 min of insulin incubation were similar to both control and ALMS1 silencing levels. human adipocytes (FIGS. 2E-F), consistent with the normal to elevated levels of AKT phosphorylation previously observed in mouse Alms1 ^foz/foz adipocytes (FIG. 1G).

The authors further investigated the dynamics of GLUT4 in human adipocytes in the absence of ALMS1. Cellular localization of the insulin receptor (IR) to the plasma membrane was not disturbed after ALMS1 silencing and was detected near the plasma membrane (PM) in the absence of insulin (Fig. 2G, upper panel). In contrast, in ALMS1-deficient adipocytes in the absence of insulin, GLUT4 lost its perinuclear localization and was detected dispersed throughout the cell cytoplasm rather than in its putative usual perinuclear localization (Fig. 2G, middle and lower panels and 2H). Upon insulin stimulation, movement of GLUT4 to PM was observed within the actin network (Fig. 2I) in both control and ALMS1-silencing adipocytes. Two hours after insulin stimulation in the absence of insulin, GLUT4 was still found dispersed throughout the cytoplasm of ALMS1-silencing adipocytes, while in control adipocytes, GLUT4 was appropriately relocalized to the perinuclear region (Fig. 2J). Since there is a balance between exocytosis and endocytosis of GLUT4 vesicles in and out of the PM, the authors tested to rule out whether defective GLUT4 endocytosis is responsible for GLUT4 missorting in ALMS1-silencing adipocytes. When examining dynamin, a key molecule of endocytosis, no differences in protein level or cellular localization were found after ALMS1 silencing in adipocytes. In addition, the average rates of labeled endosomes were similar

- 17 040194 between ALMS1 silencing and control adipocytes, arguing against an endocytosis defect causing reduced GLUT4 presence in the RM in response to insulin signaling.

Example 3 ALMS1 is required for targeted migration of TBC1D4 into PM in response to an insulin signal.

In order to understand the molecular mechanism underlying the effect of ALMS1 inactivation on GLUT4 localization, the authors determined the interaction of ALMS1 partners in human adipocytes. Immunoprecipitation (IP) using ALMS1 as a bait was performed using young mature human adipocytes (4 days after differentiation switch) followed by identification of ALMS 1 interacting partners by mass spectrometry. Among the proteins immunoprecipitated with ALMS1 was TBC1D4, a known AKT substrate GTPase required for proper retention of GLUT4 in GLUT4 sorting vesicles (GSV) and for translocation of GLUT4 to the cell membrane for intracellular glucose uptake.

Example 4 Development of a Structural Homology Model for ALMS1, TBC1D4 and aPKC.

Since the crystal structure of Alms1 has not yet been obtained, in silico structural homology modeling was used to predict the 3D structure of ALMS1 and identify structural motifs that can bind potential interacting ligands (FIG. 3a-C).

Structural model of ALMS1. The ALMS1 model was built using the fragment modeling method with the homologous modeling program, Modeller. The amino acid sequence for each exon of ALMS1 was sent to a profiled streaming algorithm available on the PISRED server, based on which suitable templates were identified. These identified template proteins were then aligned with the corresponding exon sequences and each exon was modeled separately using Modeller. Energy optimization and model selection were performed based on the energy score of the discrete optimized protein. Finally, models were assembled to build the structure of full-length ALMS1 and the full-length protein was relaxed and minimized using the NAMD molecular dynamics simulation program.

Structural model of the PTP-binding domain of TBC1D4. The PTP binding domain of TBC1D4 is within the first 160 residues. A reliable homologue structure that mimics the structure between the PTP binding domain and the Rab binding domain has not been determined. The crystal structure of the PTP domain of mouse Disabled-1 (Dab-1), 1NU2 (E-value=5.2e-17), which was identified based on the HMM pattern search in the Swiss model, was used as a template to construct the PTP binding domain TBC1D4.

The PTP domain of TBC1D4 interacts with ALMS-1. Macromolecular docking was performed using the ClusPro 2.0 algorithm. Residues located on the interaction surface with an overlap of >0.4 A were considered as interacting residues. Interproscan showed that ALMS-1 contains a WD40-like domain within the first 3871 residues. Proteins containing the WD40 domain are a family of proteins that function as scaffolds for macromolecular interactions.

The PTP binding domain of TBC1D4 interacts with ALMS1. First, the PTP-binding domain and the RabGTP-binding domain of TBC1D4 were connected to the ALMS1 model using the Cluspro 2 server to determine the most likely interaction site. Both domains were then linked to their respective interaction sites in ALMS1 using AutoDock 4.2 and their binding affinities calculated. Based on affinity, the PTP binding domain of TBC1D4 binds ALMS1 with ~100 times higher affinity than the RabGTP binding domain. Therefore, the present inventors predicted that the PTP binding domain may have a higher likelihood of interacting with the ALMS1 molecule compared to the RabGTP binding domain.

Modeling the PTP domain from TBC1D4. The phosphotyrosine binding domain in TBC1D4 was modeled after the identification of a suitable template from the Swiss Model Template Identification Algorithm.

Docking of the PTP domain and RabGTP binding domain from TBC1D4 to ALMS1. First, the PTP binding domain and the RabGTP binding domain of TBC1D4 were connected to ALMS1 using a Cluspro 2 server and an interaction site was identified. Both domains were then connected to their respective interaction sites using AutoDock 4.2 and their binding affinities calculated.

- 18 040194

Predicted numbers of ALSM-1 residues with potential for interaction with another ligand 65,66,69,72,73,74,75,76,77,78,80,87,2875,2876,2877,2878,2879,2880,2881,2882,2883,2884,2885,2887,2888, 28 89,2890,2892,2893,2894,2895,2897,2909,2910,2912,2929,2931,2932,2933,2934,2935,3557,3558,4131,144,145, 146,147,148,149,150,151,193,194,195,198,199,200,201,205,208,211,214,226,227,229,233,234,235,236,239,242 ,243,246,248,249,250,251,252,314,319,321,986,1341,1344,2269 ,113,114,115,116,123,126,127,128,1340,1438,1 439,1440,1441,1442,1443,1444,1446,1447,1448,1449,1450,1451,1452,1453,1454,1457,1458,1459,1478,1915,1 918 ,1919,1920,1922,1923,1930,2041,2042,2043,2257,2267,2483,2484,3866,218,219,220,221,222,223,224,277, 278,279,282,285,286,287,288,686,688,689,690,691,699,1856,1858,1859,1861,1862,1863,1864,1865,1866,1867 , 1868.1869.1870.1871.1872.1949.1968.1969.1971.1974.1979.1980.1981.1982.1983.1984.2104.2107.2111.2870 ,3287,793,795,796,797,1285,1314,1408,1409,1422,1423,1425,1426,1427,1430,1431, 1671,1672,1794,1797,2538,2539,2540,2555,2556,25 .2565.2567.2568.25 88.2591.2599.2603.2699, 2701.2702.3108 Projected balances ALMS1 mediating interaction with aRKS E17, D58, S59, G62, H65, L66, Q736, T737, E738, D828, S829, T1088, D1089, A1169, Q1170, F2882, L2883, E2884 Predicted numbers of arKC residues mediating interaction with ALMS1 F114, D116, C118, L121, N138, Q142, 1145, P148, G433, E545, S562, S566, F597, D601, W602, K604, E606, G620, T631, V664, 1667 Predicted TBC1D4 residues mediating interaction with ALMS1 G75, A76, R77, A78, R80, E81, V82.183 Projected balances ALMS1 mediating interaction with TBC1D4 H65, L66, S2879

The homology model showed that Almsl is expected to have an apple core structure with a large number of potential ligand binding sites centered around the core. The crystal structure of TBC1D4 was not similarly obtained and therefore we used a homologous modeling approach to predict the structure of the PTP binding domain of TBC1D4 (Fig. 3C-D). Then, in silico docking studies were performed that predicted a high binding affinity of TBC1D4 to ALMS1 via the hydrogen bonds of TBC1D4 residues G75, A76, P77, A78, R80, E81, V82, I83, which interacted with residues H65, L66, S2879 in Alms1 (Fig. .4A). Colocalization of ALMS1 and TBC1D4 was then confirmed in human adipocytes by immunofluorescence assays (FIG. 4B). Next, the expression levels of GLUT4, TBC1D4 and IRAP in ALMS1 silencing adipocytes with or without insulin stimulation were tested, but no significant differences were found (Fig. 4C). After phosphorylation with activated AKT, phosphorylated TBC1D4 (p-TBC1D4) in adipocytes targets RAB proteins such as RAB14 and RAB10 before targeting GSV migration into the PM. However, when ALMS1 silencing adipocytes were stimulated with insulin, no difference was found in the levels of TBC1D4, p-TBC1D4, RAB14 and RAB10 (FIG. 4). The authors then focused on the cellular localization of GLUT4. In the absence of insulin stimulation, TBC1D4 silencing reproduces the effect of ALMS1 silencing in mature adipocytes; impaired localization of GLUT4 throughout the cytoplasm (Fig. 4E). In response to insulin stimulation in control adipocytes, GLUT4 is released from the perinuclear region, distributed throughout the adipocyte cytoplasm (Fig. 4F), thus reproducing the distribution pattern of GLUT4 observed in adipocytes silencing ALMS1 and TBC1D4 in the absence (Fig. 4E) and presence (Fig. 4F) of insulin. The inventors further investigated the effect of ALMS1 silencing on TBC1D4 cellular dynamics in response to insulin. In the absence of insulin, TBC1D4 localizes to the perinuclear region in both control and ALMS1-silencing adipocytes (Fig. 4G), but, notably, in response to insulin, TBC1D4 was transported into the PM only in controls and not in ALMS1-silencing adipocytes. adipocytes (Fig. 4H).

Example 5 ALMS1 forms a dynamic protein complex, the ALMSome, required for insulin-stimulated glucose transport in mature human adipocytes.

Although the inventors have shown that ALMS1 silencing prevents TBC1D4 targeting in the PM, it is still unexplained whether this disruption explains the severe decrease in glucose uptake observed in ALMS1-deficient adipocytes. Therefore, the inventors compared the cellular uptake of 2-NBDG upon insulin stimulation in ALMS1 or TBC1D4-silencing or control adipocytes and found no uptake of 2-NBDG in ALMS1-silencing adipocytes compared to control adipocytes (Fig. 5A-B), while a reduced, compared to control, actual amount of 2-NBDG was still taken up by adipocytes subjected to TBC1D4 silencing (FIG. 5C). Subsequent anti-GLUT4 antibody binding assays in either ALMS1-silencing or TBC1D4-silencing adipocytes or control adipocytes after 30 min of insulin stimulation showed a high proportion of GLUT4 in PM in control adipocytes and in TBC1D4-silencing adipocytes, but not ALMS1-silencing cells (Fig. 5D-F), indicating that a secondary defect in TBC1D4 targeted migration into PM in Alms1-silencing cells does not alone explain the very severe defect in glucose transport and GLUT4 PM expression in ALMS1-deficient cells. Further examination of Alms1 IP data revealed several ATPase proton (H ⁺ ) pump type V subunits (A, B, D1 and G2), which, as the authors then showed, were expressed in mature adipocytes (Fig. 5G) together with aPKC activating kinase H ⁺ -pumps under con

- 19 040194 insulin control. Using the in situ PLA Duolink method targeting ALMS1 and the A1 and D1 subunits of the proton pumps (Fig. 5H), as well as co-immunostaining with ALMS1, A1 and D1 of the V-ATPase and aPKC subunits (Fig. 5H, I), the present authors inventions confirmed that ALMS1 was in close proximity to the V-ATPase H ⁺ pumps in mature adipocytes in the presence of insulin. ALMS1 is colocalized with the V0D1 subunit of the proton pump (Fig. 5I), which is embedded in the GSV membrane, indicating that in the adipocyte ALMS1 is transported along with proton pumps localized in GSV. Using a structural in silico model of partners interacting with ALMS1, the authors determined the motif of PKC binding to Alms1. The binding sites for TBC1D4 and aPKC on Alms1 were so close that the model showed that simultaneous attachment of both proteins on Alms1 was not possible due to steric hindrance (Fig. 5J). As a result, the authors put forward a hypothesis. To test their hypothesis that in adipocytes, ALMS1 binding to aPKC, or otherwise TBC1D4, is under inverse control of the insulin signaling pathway, the authors performed additional IPs using ALMS1 as a bait, but this time using mature human adipocytes. cultured in the presence or absence of insulin, while conducting IP immunoblotting for both aPKC and TBC1D4. The results showed that aPKC was only precipitated by Alms1 and detected by immunoblotting in insulin-free adipocyte extracts (Fig. 5K), while TBC1D4 was precipitated by ALMS1 and detected in insulin-incubated adipocyte extracts, in accordance with the author's reciprocal insulin model. regulated Alms1 binding (Fig. 5L).

Example 6 ALMSoma is required for acidification of GSV prior to delivery of GLUT4 to the plasma membrane.

Although AKT phosphorylation of TBC1D4 has been known to result in targeted GLUT4 migration in some cases, the final step of GSV-PM fusion is an insulin-regulated AKT-independent event that requires osmotic swelling of GSV by vATPase H ⁺ pumps. However, there was no information on the actual signal and mechanism of activation of H ⁺ pumps by insulin. The authors tested the possibility of preventing acidification of GSV upon inactivation of ALMS1 and, as a consequence, the chemo-osmotic release of GLUT4 in PM using the acidotrophic dye acridine orange, which emits green fluorescence at low concentrations and orange-red fluorescence at high concentrations in lysosomes in which acridine orange is protonated and sequestered. In the absence of insulin, no orange-red fluorescence was detected in adipocytes. In contrast, insulin induced a rapid red color in control mature human adipocytes (FIG. 6A) but not in ALMS1-silencing adipocytes (FIG. 6B), indicating a loss of insulin-mediated acidification of lysosomes in ALMS1-silencing adipocytes.

The inventors then tested whether ALMS1-silencing adipocyte acidification with nigericin (NIG), an electrically neutral K ⁺ /H ⁺ exchange ionophore known to induce osmotic swelling of GSV, could bypass the Alms1-associated GLUT4 fusion defect and glucose uptake. . NIG treatment resulted in rapid acidification of both control and ALMS1 silencing adipocytes (Fig. 6C-D), resulting in activation of swelling and fusion of intracellular vesicles. In parallel, electron microscopy analysis showed vesicles adjacent to the PM without fusion in the absence of insulin in both control and ALMS1 silencing adipocytes (Fig. 6E-F, upper panels). Insulin treatment induced vesicle swelling (FIG. 6E, middle panels) associated with vesicle fusion with PM for glucose uptake only in control adipocytes (FIG. 6E, middle panels), but not in ALMS1-silencing adipocytes (FIG. 6F, middle panels). However, NIG induced vesicular swelling and PM fusion in both control and ALMS1 silencing adipocytes (Fig. 6E-F, lower panels). NIG treatment restored glucose uptake in ALMS1 silencing adipocytes. While insulin had little effect on the stimulation of 2-NBDG uptake in ALMS1 silencing adipocytes (Fig. 6G-H, upper and middle panels), NIG not only restored vesicle fusion, but was also shown to restore uptake 2 -NBDG in ALMS1 silencing adipocytes to levels observed in control cells (FIGS. 6G-H, lower panels).

This restored glucose transport in NIG-treated ALMS1-silencing adipocytes correlated with repaired GLUT4 fusion to PM (FIG. 7A), but not TBC1D4 targeting migration to PM (FIG. 7B); and resulted in the restoration of TG-filling of adipocytes subjected to ALMS1 silencing 24 h after NIG treatment.

Example 7 Identification of peptide inhibitors of PKC binding to Alms1.

Once the site of the binding interaction between two proteins became known, it became possible to apply information about the conformation and amino acids of each protein involved in mediating this interaction in computer models to develop peptides or small molecular weight drugs that, when bound to the site of interaction, are capable of sterilizing

- 20 040194 physically or otherwise prevent the bonding interaction. Thus, the authors sought to identify peptides that inhibit the interaction of ALMS1 and aPKC or TBC1D4 using their previously described in example 4 structural models of ALMS1, TBC1D4 and aPKC. Peptides predicted by this method to block the interaction between aPKC and ALMS1 include the following sequences: LDSDSHYGPQHLESIDD (SEQ ID NO: 5), DSHQTEETL (SEQ ID NO: 6), QQTLPESHLP (SEQ ID NO: 7), QALLDSHLPE (SEQ ID NO: 7), QALLDSHLPE (SEQ ID NO: : 8). PADQMTDTP (SEQ ID NO: 9), HIPEEAQKVSAV (SEQ ID NO: 10) or SCIFLEQ (SEQ ID NO: 11). The peptide identified by this method to block the interaction between TBC1D4 and ALMS1 had the sequence GCGAPAAREVILVL (SEQ ID NO: 12).

Example 8 Expression of aPKC specific ALMS1-interacting domain in mature adipocytes induces glucose uptake in the absence of insulin.

Next, the authors tested the hypothesis that insulin mediates the release of aPKC from the ALMSoma complex to induce glucose uptake. To do this, they cloned the aPKC interacting domain (SEQ ID NOs: 14 and 15) into a lentiviral vector along with a Flag tag. The selected sequence was the minimal aPKC sequence (min-aPKC-FLAG) without steric hindrance to the TBC1D4-ALMS1 interaction site. Adipocyte-expressed minaPKC-FLAG competes with endogenous aPKC, preventing its binding to the ALMSoma and thereby favoring insulin-mediated binding of TBC1D4 to the ALMSoma. Next, mature adipocytes were infected with either control or min-aPKC-lentiviral particles to evaluate the effect of min-aPKC-FLAG on glucose uptake. 48 hours after infection, min-aPKC-FLAG was immunodetected using an anti-FLAG tag antibody (FIG. 9). For in vitro evidence, we treated mature adipocytes as described (FIG. 9) and then incubated treated mature adipocytes with 2-NBDG to evaluate the effect of min-aPKC-FLAG on glucose uptake. Interestingly, 2NBDG was taken up in min-aPKC-FLAG-treated adipocytes in the absence of INS (FIG. 8A, left column), corresponding to a 3.5-fold increase over control (FIG. 8B). On the other hand, no significant difference was observed in the presence of INS (Fig. 8A, right column and 8B). These data suggest that targeting the interaction of ALMS1 and aPKC is sufficient to induce glucose uptake in adipocytes, regardless of the presence of INS.

Obtaining a lentiviral vector carrying the aPKC domain.

The ALMS1 interacting domain of human PKCa was amplified from N-terminal FLAG-tagged human HEK293 cDNA using forward 5'-3'gtacGAATTCGCCACCATGGATTACAAGGATGACGACGATAAGCTCACGGACTTCAATTTCCTC (SEQ ID NO: 16) and reverse 5'-tagcGGATCCT NOCATACTGCACTCTGTAAGATGGG-3' (SEQ ID NO: 16) and reverse 5'-tagcGGATCCT NOCATACTGCACTCTGTAAGATGGG-3' (SEQ ID NO: 17 ) primers and cloned into the PCDH-EF1-MCS-IRES-Puro lentiviral vector (System Biosciences). To obtain the virus, PKCa-lentiviral vectors were transfected into 293TN cells (System Biosciences) together with the packaging plasmids psPAX2 and pMD2. G (Addgene) with a mass ratio of 3:2:1, respectively, and using Lipofectamine 2000 (Life Technologies). 48 hours after transfection, the culture supernatant was collected by centrifugation at 500xg for 10 min followed by filtration through a 0.45 μm syringe filter with PES membrane (Sartorius). The virus solution was then concentrated by adding 1/2 volume of cold 30% (w/v) PEG6000 dissolved in 0.5M NaCl and incubated overnight at 4°C with occasional stirring. The mixture was then centrifuged at 3000xg for 15 min at 4°C. The pellet containing the lentiviral particles was then resuspended in 1 ml DMEM medium and stored at -80°C until the target cells were infected.

Claims (14)

CLAIM 1. Use of a molecule preventing the binding of aPKC (α-type protein kinase C) to ALMS1 (Ahlström syndrome protein 1) in the treatment or delay of the onset or onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance , hyperglycemia, obesity and hyperinsulinemia, where the molecule is selected from the group consisting of peptides or polypeptides or peptidomimetics, antibodies, fragments or derivatives thereof, aptamers, spiegelmers and chemical compounds. 2. Use according to claim 1, characterized in that the molecule is intended for use in the treatment or delay in the development or onset of type 2 diabetes mellitus. 3. Use according to claim 1 or 2, characterized in that the molecule does not interfere with the binding of TBC1D4 to ALMS1. 4. Use according to any one of claims 1 to 3, characterized in that the molecule is a peptide of less than 50 amino acids, preferably less than 20 amino acids. 5. Use according to claim 4, characterized in that the molecule is a peptide containing - 21 040194 amino acid sequence of the ALMS1 fragment (SEQ ID NO: 1). 6. The use according to claim 5, characterized in that the molecule is a peptide containing the amino acid sequence of the ALMS1 fragment, including one or more of the residues that are predicted to mediate interaction with aPKC, in particular one or more of the residues selected from a list consisting of E17, D58, S59, G62, H65, L66, Q736, T737, E738, D828, S829, T1088, D1089, A1169, Q1170, F2882, L2883 and E2884. 7. Use according to claim 5 or 6, characterized in that the molecule is a peptide containing or consisting of one of the following sequences: LDSDSHYGPQHLESIDD (SEQ ID NO: 5); DSHQTEETL (SEQ ID NO: 6); QQTLPESHLP (SEQ ID NO: 7); QALLDSHLPE (SEQ ID NO: 8); PADQMTDTP (SEQ ID NO: 9); HIPEEAQKVSAV (SEQ ID NO: 10); SCIFLEQ (SEQ ID NO: 11) and a fragment containing 6 contiguous amino acids. 8. Use according to claim 4, characterized in that the molecule is a peptide containing the amino acid sequence of the aPKC fragment (SEQ ID NO: 4). 9. The use according to claim 8, characterized in that the molecule is a peptide containing the amino acid sequence of the aPKC fragment, including one or more of the residues predicted to mediate interaction with ALMS1, in particular one or more of the residues, selected from a list consisting of F114, D116, C118, L121, N138, Q142, I145, P148, G433, E545, S562, S566, F597, D601, W602, K604, E606, G620, T631, V664 and I667. 10. An in vitro or ex vivo method for identifying molecules suitable for treating or slowing the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity, and hyperinsulinemia, wherein the method comprises the steps of : (a) analysis of the ability of the candidate molecule to prevent the binding of aPKC to ALMS1; and (b) selecting molecules capable of preventing the binding of aPKC to ALMS1 for use in treating or slowing the progression or delaying the onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity, and hyperinsulinemia . 11. The method of claim 10, wherein the method further comprises testing the ability of the selected molecule to interfere with TBC1D4 binding to ALMS1 and selecting molecules that do not interfere with binding. 12. The method according to claim 10 or 11, characterized in that the binding is determined in the cellular system in response to insulin. 13. The method according to any one of claims 10 to 12, characterized in that the binding is determined in the presence and/or absence of insulin. 14. The use of a molecule that enhances the binding of TBC1D4 to ALMS1 for the treatment or delay of the development or occurrence of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, hyperglycemia, obesity and hyperinsulinemia, where the molecule is selected from the group, consisting of peptides or polypeptides or peptidomimetics, antibodies, their fragments or derivatives, aptamers, spiegelmers and chemical compounds.